The link between stress and alcohol drinking is complex, but some crucial neuroadaptations in stress pathways are beginning to be understood. The current proposal explores the neural substrates of how early life stress renders mice vulnerable to excessive alcohol drinking in adulthood. One interesting neuropeptide that can be disrupted is the stress neuropeptide corticotropin-releasing factor (CRF) in the brain. CRF may be an important regulatory hormone in social alcohol drinkers, but it is the dysfunction of CRF that may develop during alcohol dependence. CRF can be found in distinct brain structures, like the ventral tegmental area (VTA). The VTA is one site that can signal for the rewarding properties of drugs and communicate stress through the neurotransmitter dopamine (DA). The dysregulation of the CRF network may be crucial to influencing alcohol drinking driven by stress. To critically examine these theories, researchers can use maternal separation stress (MSS) in mice to induce neuroadaptations to increase alcohol drinking in adulthood. In the first set of experiments, mouse pups are separated from maternal care for 3 hours per day for 2 weeks. Later in adulthood, mice are given intermittent access to alcohol, an advantageous method to test escalated voluntary alcohol drinking. We will then measure if pharmacological treatment with CRF-R1 antagonists into the VTA can prevent intensified stress-altered DA impulse flow. Changes in DA as a result of alcohol drinking are measured with microdialysis in the prefrontal cortex (PFC), a terminal region of the VTA, in stressed mice vs. unstressed mice. CRF protein in the brain will also be measured after MSS and after escalated alcohol drinking. In a second group of experiments, researchers test if genetic prevention of forebrain CRF-R1 can influence alcohol drinking. Mice lacking CRF-R1 gene transcript in the forebrain also undergo MSS, alcohol drinking, and dialysis to assess DA output. Ultimately, these methods allow for site-specific manipulations to study CRF modulation of the VTA-PFC pathway in stress-escalated behavior. The pharmacological and genetic approaches not only complement each other, but also allow for considerable training potential. Altogether, identifying the crucial pathways in stress-escalated drinking would advance scientific understanding of the mechanisms behind the transition to alcoholism. Findings from the proposed experiments may reveal those interactions between genes and the social environment that differentiates social use from excessive drinking. Ultimately, targeted treatment for alcohol use disorders may include pharmacotherapy and management of early life stress.